Abstract

It has been proposed that all classes of nucleic acid polymerases use the same two‐metal‐ion mechanism for nucleotide incorporation. The main chemical kinetic scheme is that the oxygen atom of the
‐OH group of the transcript primer, acting as a nucleophile, forms a phosphodiester bond with the first phosphate of the (deoxy)nucleoside triphosphate and the other two phosphates form a pyrophosphate leaving group. While some molecular modeling studies on DNA polymerases have been performed to investigate the detailed chemical steps involved in the kinetic scheme, few theoretical studies on RNA polymerases are available in the literature. Here, we report a molecular dynamics study of nucleotidyl transfer catalyzed by the yeast RNA polymerase II, which is based on the most recently published crystal structures. We particularly focus on the creation of the nucleophile, i.e., deprotonation of the
‐OH group. Two pathways are examined: (i) proton transfer to the conserved Asp485 residue of the active site in association with nucleophilic attack and (ii) proton transfer to a nearby water molecule before nucleophilic attack. All the molecular dynamics simulations are carried out by a recently developed reactive force field, ReaxFF, whose parameters are derived directly from quantum chemical calculations. The rate‐limiting step of the reaction in both cases is the dissociation of the pyrophosphate leaving group, which needs about 23 kcal/mol of activation energy. The nucleophilic attack needs about 19 kcal/mol of activation energy for pathway (i) and 17 kcal/mol of activation energy for pathway (ii). The water‐assisting deprotonation just needs about 7 kcal/mol of activation energy. These data indicate that pathway (i) is comparable to pathway (ii). If water misses the deprotonation of the
‐OH group before nucleophilic attack in the latter pathway, the general base (Asp485) of the polymerase active site can readily perform the deprotonation in the attack through the former pathway.